PROFESSIONAL DEVELOPMENT

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1 PROFESSIONAL DEVELOPMENT AP Biology Cell-to-Cell Communication Cell Signaling Special Focus

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3 Contents 1. Introduction...1 Julia Kay Christensen Eichman 2. Introductory Lab: Cell-to-Cell...3 Julia Kay Christensen Eichman 3. Cell Linkages: Integrins Elizabeth A. Cowles 4. AP Biology Free-Response Questions and Scoring Rubrics...21 Julia Kay Christensen Eichman 5. Additional Web Resources...47 Carolyn Schofield Bronston 6. About the Editor About the Authors iii

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5 Introduction Julia Kay Christensen Eichman Missouri Southern State University Joplin, Missouri Cell-to-cell communication, or signaling, is an important part of understanding cell functions as well as system functions. There are several types of signaling, such as neurotransmitters that are recognized in the synapse, antigens triggering antibody responses, and target cells responding to specific hormones. This project provides more information about the signaling process using integrins as the mechanism. The document opens with a very effective introductory lab focused on cellto-cell communication. Following this lab, author Elizabeth Cowles elaborates on cell linkages and integrins in an attempt to offer students and teachers additional background information and practical applications. These materials also include appropriate AP Biology Exam free-response questions and their rubrics from previous years, as well as informative and interactive Web sites. These resources provide teachers with additional information regarding cell communication as well as animated examples of other types of signaling. If access allows, teachers may use the activities and information presented on these Web sites to introduce, develop, and reinforce concepts associated with cell communication. Likewise, teachers may use the free-response questions to not only reinforce the concepts embedded within cell communication, but also to aid in the understanding of this communication. These questions also serve to test students analytical and reasoning skills while reflecting appropriate lab experiences they should possess.

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7 Introductory Lab: Cell-to-Cell Julia Kay Christensen Eichman Missouri Southern State University Joplin, Missouri Is there an exchange of chemical information between cells? Purpose To determine if one cell has an effect on the conditions in an adjacent cell. This is a simple representation of cell-to-cell transfer of information with diffusion through two cell membranes. Materials One eight-inch by four-inch plastic disposable storage box with a lid (clear or translucent without color) Four pieces of one-inch by eight-inch dialysis tubing that have been soaked overnight 1 percent starch solution Diluted Lugol s solution (4 ml per 200 ml of distilled water) String or dialysis tubing clamps Distilled water Procedure Clamp or tie off one end of each of the dialysis tubing. Place enough distilled water in the plastic storage box to cover the bottom. Fill two pieces of the dialysis tubing with 1 percent starch solution and seal the open ends.

8 Special Focus: Cell-to-Cell CommunicationCell Signaling Fill the remaining two pieces of dialysis tubing with the diluted Lugol s solution and seal the open ends. Place the completed tubing into the plastic storage box, alternating first with Lugol s-filled tubing and then a starched-filled tube, then another Lugol s-filled tube, and finally the last starch-filled tube. The tubes should fit snugly into the box so the sides of the tubing are in complete contact. Place the lid on the plastic storage box to help keep the tubing moist, just as cells are moist at all times. Make observations every five minutes to observe any changes in the tubes. Are there any color changes? Analysis 1) Explain why it was important to keep the system moist. 2) Were there color changes in any of the tubes? If so, what do these changes indicate? 3) Compare and contrast the dialysis tubing bags in contact with each other to cells that are in contact with each other. 4) The dialysis tubing bags serve as a model for a community of living cells. In what ways is the model an accurate portrayal of cell systems and in what ways is it flawed? 5) Describe two specific examples of cell-to-cell communication, naming the type of cell and what chemical message is passed. Lab Questions Answer Key Question 1: The system must be kept moist for the solutions to diffuse across the dialysis tubing (membranes) just as the cell membranes are moist for the same defusing actions to take place. Question 2: The starch tube will initially appear clear to milky white, depending on the amount of starch; the iodine tube will initially be copper colored. The starch tube will turn dark 4

9 Introductory Lab: Cell-to-Cell as the iodine diffuses into it. The iodine tube will become lighter in color as the iodine leaves. Question 3: The dialysis tubes lying side by side are similar to cell membranes because they are moist and they allow small particles to diffuse. They are different because the only method of material passage is diffusion. Question 4: Cell membranes have protein channels and allow materials to be moved by endocytosis and exocytosis, and they are moist to allow for diffusion. The dialysis tubing is a model for the diffusion portion of the cell activity, and it also demonstrates the need for the system to be moist. Question 5: Examples include neurotransmitters in the synapse, antigens triggering antibody response, target cells responding to specific hormones, and many others. 5

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11 Cell Linkages: Integrins Elizabeth A. Cowles, Ph.D. Eastern Connecticut State University Willimantic, Connecticut Introduction Communication is the name of the game, especially in cells; specialized receptors called integrins provide vital communication links between the interior and exterior of the cell. Integrins are transmembrane proteins that act as mechanotransducers and signal conductors, providing a physical link between the extracellular matrix (ECM) and the cell s cytoskeleton. Although integrins do not have intrinsic enzymatic activity, they can interact with enzymes such as kinases that have specific signaling functions. Integrins are involved in many cellular processes, such as differentiation, migration, proliferation, ECM protein expression, activation of growth factors, apoptosis, and cell survival. Interestingly, integrins work from either direction: They can bind to extracellular ligands, thus triggering intracellular signal cascades, or they can be activated by factors from within the cell to influence the relationship of the cell with its environment. Integrins are heterodimers, meaning that they are composed of two distinct subunits termed α and β. Humans produce 18 different alpha chains (the larger subunit weighing kda) and 8 different betas (smaller subunits weighing kda), which combine to form different integrins. So far, 24 human integrins have been identified, each named for their two-component subunits (α2β1, for example). Studies of invertebrate species such as Drosophila melanogaster and Caenorhabditis elegans have revealed the presence of integrins (though there are fewer varieties), and the basic heterodimer structure is highly conserved among all animals. Proteins very similar to integrins are found in plants, fungi, or prokaryotes; these proteins may be important in touch (thigmo) responses in these organisms

12 Special Focus: Cell-to-Cell CommunicationCell Signaling (Jaffe et al. 2001), in gravity perception in plants (Katembe et al. 1997), and in the binding of a pathogenic fungus to fibronectin (Kottom et al. 2008). Integrins typically span the cell s plasma membrane with the N- or aminoterminus of both subunits extending into the extracellular matrix, providing potential ligand binding sites. The subunits interact with each other and exist in either a low-affinity or a high-affinity conformation depending on external and internal signals (Humphries and Liddington 2002). The integrins are normally bent in the low-affinity state, but open like a switchblade upon activation via phosphorylation of the β subunit s cytoplasmic end. Figure 1 A model of integrin activation. Schematic representation of a heterodimeric integrin molecule in the bent, inactive conformation (left) and the upright, active conformation (right). The switch in conformation is triggered by the binding of a protein, in this case talin, to the small cytoplasmic domain of the β subunit. The binding of talin induces a separation of the two subunits and conversion to the active conformation. Activated integrins typically become clustered as the result of interactions of their cytoplasmic domains with the underlying cytoskeleton. The extracellular ligand, in this case a collagen fiber, binds between the two subunits in the head region of the activated integrin dimer. Figures 1 4 from Cell and Molecular Biology: Concepts and Experiments by Gerald Karp. New York: John Wiley & Sons, Inc., Used with permission of John Wiley & Sons, Inc. This physical change modulates both the integrin s affinity for its ligand and also the stability of the attachment. The relatively small amino acid subunit ends that extend into the cytoplasm allow interactions with intracellular proteins; the β4 cytoplasmic tail is so long, it can even bind to intermediate filaments in the cytoskeleton. Fibronectin, which has important cell migration and wound healing functions, provides a good example of extracellular protein interaction with integrins. Fibronectin contains an RGD or arginine glycine aspartate sequence, which is recognized by the integrin α5β1. The integrin simultaneously interacts with talin, which provides a physical link to actin filaments, thus allowing cells to migrate along the fibronectin. 8

13 Cell Linkages: Integrins Figure 2 Focal adhesions are sites where cells adhere to their substratum. This drawing of a focal adhesion shows the interactions of integrin molecules with other proteins on both sides of the lipid bilayer. The binding of extracellular ligands, such as collagen and fibronectin, is thought to induce conformational changes in the cytoplasmic domains of the integrins that cause the integrins to become linked to actin filaments in the cytoskeleton. Linkages with the cytoskeleton are mediated by various actinbinding proteins, such as talin and a-actinin, that bind to the β subunit of the integrin. The cytoplasmic domains of integrins are also associated with protein kinases, such as FAK (focal adhesion kianse) and Src. The attachment of the integrin to an extracellular ligand can activate these protein kinases and start a chain reaction that transmits signals throughout the cell. The association of myosin molecules with the actin filaments can generate traction forces that are transmitted to sites of cell substrate attachment. Other proteins containing the RGD sequence include vitronectin, bone sialoprotein, von Willebrand factor, fibrillin, fibrinogen, thrombospondin, PECAM (platelet endothelial cell adhesion molecule), tenascin, and LAP-TGFβ (latency associated peptide transforming growth factor-β). The RGD motif is also used for interactions between ECM proteins and integrins αvβ1 and α8β1. Other integrins use a different tripeptide sequence, leucine-aspartate-valine (LDV), to recognize and bind proteins important for intercellular adhesion, such as VCAM (vascular cell adhesion molecule), ICAM (intercellular adhesion molecule), factor X, mucosal adhesion cell adhesion molecule (MadCAM), and E-cadherin. Integrins containing the α1 and α2 subunits have an A domain, similar to von Willebrand factor, a blood glycoprotein important in clotting. These subunits combine with β1 to form receptors for collagen, thrombospondin, and laminin. Integrins and Cellular Signaling Inactive integrins are dispersed over the cell surface. Upon binding to ECM proteins, integrins migrate within the cell membrane to cluster and form focal adhesion sites in a process called activation. These sites may then include interactions with several additional proteins such as focal adhesion kinase (FAK), paxillin, talin, and tensin (Tadokoro et al. 2003; Lo 2006). Integrin interactions between talin, vinculin, α-actinin, and paxillin provide a physical linkage between the ECM and the actin cytoskeleton; these links are critical 9

14 Special Focus: Cell-to-Cell CommunicationCell Signaling for cell anchorage and migration. Phosphorylation of the β tail, in the cytoplasm, disrupts the talin integrin interaction, thus permitting cell movement. Integrin activation via external factors can set off a cascade of events; this is called outside-in signaling. These interactions can involve several proteins, in which the integrin has a critical mediating role. Outside-in signaling can result in modification of the cytoskeleton, cause cellular proliferation or migration, and determine cell survival or apoptosis. One example is the MAPK/ERK (mitogenactivated protein kinase or extracellular signal-related kinase) signal pathway, which is turned on by integrin-extracellular ligand interactions. Figure 3 This pathway controls gene expression by increasing the stability of c-jun and increasing transcription of the protein c-fos. C-jun and c-fos combine to form AP-1 (activating protein-1), which binds to specific DNA promoters; therefore, integrins can control gene transcription via the MAPK/ ERK signaling kinases. In particular, AP-1 induces expression of integrin α2β1 and its ligand, collagen. Another example of integrin-mediated control of transcription involves activation of the α5β1 integrin, which increases ERK; this ultimately up-regulates the transcription of the protein Bcl-2, which is an anti-apoptotic signal. High Bcl-2 levels prevent apoptosis, or programmed cell death. Temporary loss of integrin-substrate contact is necessary for migration; however, loss of adhesion may trigger apoptosis. This balance between integrin binding and apoptosis prevents inappropriate cell migration and adhesion; this equilibrium is often aberrant in cancer cells. Signaling via integrins is not always outside-in, nor is it always unidirectional. 10

15 Cell Linkages: Integrins Figure 4 Blood clots form when platelets adhere to one another through fibrinogen bridges that bind to platelet integrins. The presence of synthetic RGD peptides can inhibit blood clot formation by competing with fibrinogen molecules for the RGD-binding sites on platelet αiiiβ3 integrins. Nonpeptide RDG analogs and anti-integrin antibodies can act in a similar way to prevent clot formation in high-risk patients. Recent research has focused on the intracellular Rho family of GTPases. Rho coordinates cell functions such as cell adhesion, cell migration, gene expression, and the cell cycle. Integrins activated by extracellular factors regulate Rho through the MAPK/ERK pathway. Rho, however, increases integrin avidity ( stickiness ) and the numbers of stress fibers, which in turn regulates the formation of integrin focal adhesion sites (Schwartz and Shattil 2000; Lo 2006). Therefore, an internal molecule like Rho can modulate integrin ECM interactions by changing the integrin conformation to the active form. The inside-out signaling is exemplified by the blood clotting system. Platelets have inactive integrins, which prevent inappropriate adhesion to vessel walls (Horowitz 1997). Damage to endothelial cells exposes thrombin, which activates platelets that come in direct contact by causing their αiibβ3 integrin to become more adhesive and to bind fibrinogen. Platelets bind to the thrombin without the assistance of integrins; the thrombin platelet interaction elicits intracellular signals, which change integrin adhesiveness. 11

16 Special Focus: Cell-to-Cell CommunicationCell Signaling Figure 5 From Biology, by Robert J. Brooker, Eric P. Widmaier, Linda Graham, and Peter Stiling. New York: McGraw-Hill Science, Used with permission of The McGraw-Hill Companies. The now-activated integrins on the platelet surfaces capture circulating von Willebrand factor and fibrinogen, which form the blood clot. Glanzmann s thrombasthenia is caused by a mutation in either the αiib or β3 integrin genes; the disorder is characterized by abnormal bruising and bleeding and is often mistaken for hemophilia. Antagonists (inhibitors) of αiibβ3 integrin, such as abciximab and xemilofiban, are used to reduce clot formation in patients at high risk for stroke or heart attacks. Leukocytes infiltrate damaged tissues, but only do so when their αmβ2 or αlβ2 integrins are activated by cytokines via inside-out signaling (Horowitz 1997). Cytokines are small, secreted proteins which regulate immunity and inflammation. The integrins mediate attachment to vascular endothelial ICAMs (intercellular adhesion molecules), and then the leukocyte migrates between the endothelial cells. Leukocyte adhesion deficiency (LAD), a very rare human disorder in which patients lack β2 or make defective β2, occurs when phagocytes cannot attach to endothelial cells. LAD patients often succumb to bacterial infections early in life. 12

17 Cell Linkages: Integrins Many integrin signaling events are coupled with growth factor responses; this is because cellular responses, such as migration and mitosis, require integrin-substrate interactions or anchorage (Giancotti and Ruoslahti 1999). For example, the platelet-derived growth factor (PDGF) receptor responds maximally only when αvβ3 is bound to the ECM and PDGF increases the amount of αvβ3 in the plasma membrane. This synergistic activity between the PDGF receptor and the integrin increases cell migration and wound healing. The interactions between the growth factor and integrin signaling pathways fine-tune the cell s response to its external environment and allow for coordinated regulation. Integrins in Health and Disease Angiogenesis, or the production of new blood vessels, is critical not only during development but also during oncogenesis or cancer development; tumors need a ready blood supply for survival. αvβ3 integrin attachment to the ECM is regulated by the vascular endothelial growth factor (VEGF) receptor; impairing the VEGF receptor integrin interaction reduces angiogenesis (Mahabeleshwar et al. 2006). Drugs that interfere with integrin function during angiogenesis are in clinical trials; Vitaxin, an anti-αvβ3 antibody, shrinks tumors by decreasing blood vessels and Avastin, an anti- VEGF monoclonal antibody, is used for treating colorectal cancers. Researchers have noted that certain breast cancers metastasized to bone. It appears that αvβ3 expression is elevated in breast cancer tissue, and this integrin recognizes bone sialoprotein, a major bone matrix constituent. Cancer cells migrating from breast tumors bind to bone tissue through αvβ3 and become established (Sloan et al. 2006). Scientists are investigating whether αvβ3 antagonists could prevent metastases (Zhao et al. 2007). Unpublished data from Barbara Susini s laboratory (University of California, San Diego) indicate that an α4β1 antagonist inhibits lymph node blood vessel development; because breast cancer spreads via the lymphatic system, such an antagonist may prevent breast cancer metastases. Viral and bacterial pathogens take full advantage of integrins. Bacterial pathogens use integrins to maintain contact and prevent removal from the host, and then gain entry (Scibelli et al. 2007). Binding allows the bacteria to be phagocytosed, to inject virulence factors, or to adhere indirectly through fibronectin. The α5β1 integrin that binds fibronectin is recognized by Shigella; several Shigella protein antigens mediate bacterial-directed endocytosis. Staphlococcus aureus and Streptococcus spp. express fibronectin-binding proteins, and indirectly attach to cells through the ECM. 13

18 Special Focus: Cell-to-Cell CommunicationCell Signaling Viruses also employ integrins to bind and gain entry into cells (Stewart and Nemerow 2007). Adenovirus is recognized by the αv integrin. The herpes virus outer envelope glycoprotein has a sequence that mimics a metalloprotease; this glycoprotein binds to β1 and αvβ3 integrins. Hantaviruses, which directly affect platelet and endothelial cell functions, have αiibβ3 and αvβ3 as receptors. Binding not only allows the virus access to the cell s synthetic machinery but also to its signaling pathways. Novel vaccines, therapeutic strategies, and antiviral drugs may be based upon integrin antagonists. A new area of integrin research is in nanoscale technology and material science (Stevens and George 2005). Implants often fail because cells do not attach and reproduce on the foreign surface. Titanium implants are commonly used in dental and joint replacements, but do not readily support osteogenesis. Implants coated with matrices of calcium carbonate (hydroxylapatite) and RGD-containing proteins, such as fibronectin, collagen or related peptides, bond bone-forming osteoblasts better than uncoated material (Reyes et al. 2007). These coatings promoted α2β1 binding, which triggered the expression of osteoblast-specific genes, which resulted in increased cell differentiation and bone production. Integrins are involved in many cellular and organismal processes, including tissue remodeling, immunology, and proliferation. These biological activities require coordination between the internal and external cellular environments; integrins provide some of the critical communication links. Insights into these fascinating cell surface receptors will help us design new therapies for cancer, inflammation, and pathogen-related diseases. Bibliography Giancotti, F. G., and E. Ruoslahti. Integrin Signaling. Science 285 (1999): Horowitz, A. F. Integrins and Health. Scientific American, May 1997; Humphries, M. J., and R. C. Liddington. Molecular Basis of Integrin-Dependent Cell Adhesion in Protein-Protein Recognition, In Frontiers in Molecular Biology: Protein-Protein Recognition, edited by C. Kleanthous, New York: Oxford University Press,

19 Cell Linkages: Integrins Hynes, R. O. Integrins: Bidirectional, Allosteric Signaling Machines. Cell 110 (2002): Jaffe, M. J., A. C. Leopold, and R. C. Staples. Thigmo Responses in Plants and Fungi. American Journal of Botany 89 (2002): Katembe, W. J., L. J. Swatzell, C. A. Markaroff, and J. Z. Kiss. Immunolocalization of Integrin-Like Proteins in Arabidopsis and Char. Physiologia Plantarum 99(1) (1997), Kottom, T. J., C. C. Kennedy, and A. H. Limper. Pneumocystis PCINT1, a Molecule with Integrin-Like Features That Mediates Organism Adhesion to Fibronectin. Molecular Microbiology (online early articles) (2008), 67: Lo, S. H. Focal Adhesions: What s New Inside. Developmental Biology 294 (2006): Mahabeleshwar, G. H., W. Feng, D. R. Phillips, and T. V. Boyzova. Integrin Signaling Is Critical for Pathological Angiogenesis. Journal of Experimental Medicine. 203(11) (2006): 2,495 2,507. Reyes, C. D., T. A. Petrie, K. L. Burns, Z. Schwartz, and A. J. Garcia. Biomolecular Surface Coating to Enhance Orthopaedic Tissue Healing and Integration. Biomaterials 28(21) (2007): 3,228 3,238. Schwartz, M. A., and A. J. Shattil. Signaling Networks Linking Integrins and Rho Family GTPases. Trends in Biochemical Sciences 25 (2000): Scibelli, A., S. Roperto, L. Manna, L. M. Pavone, S. Tafuri, R. D. Morte, and N. Staiano. Engagement of Integrins as a Route of Invasion by Bacterial Pathogens. Veterinary Journal 173 (2007): Sloan, E. K., N. Pouliot, K. L. Stanley, J. Chia, J. M. Mosley, D. K. Hards, and R. L. Anderson. Tumor-Specific Expression of αvβ3 Integrin Promotes Spontaneous Metastasis of Breast Cancer to Bone. Breast Cancer Research 8 (2006): R20. Stevens, M. M., and J. H. George. Exploring and Engineering the Cell Surface Interface. Science 310 (2005):

20 Special Focus: Cell-to-Cell CommunicationCell Signaling Stewart, P. L., and G. R. Nemerow. Cell Integrins: Commonly Used Receptors for Viral Pathogens. Trends in Microbiology 15(11) (2007): Tadokoro, S., S. J. Shattil, K. Eto, V. Tai, R. C. Liddington, J. M. de Pereda, M. H. Ginsberg, and D.A. Calderwood. Talin Binding to Integrin β Tails: A Final Common Step in Integrin Activation. Science 302 (2003): Zhao, Y., R. Bachelier, I. Treilleux, P. Pujuquet, O. Peyuchaud, R. Baron, P. Clément- Lacroix, and P. Clézardin. Tumor αvβ3 Integrin is a Therapeutic Target for Breast Cancer Metastases. Cancer Research 67(12) (2007): 5,821 5,

21 Integrin Definitions Adenovirus: Double-stranded DNA virus; causes respiratory tract infections. α-actinin: Actin-crosslinking protein; found in the cytoskeleton. AP-1: Activator protein-1 transcription factor; dimeric complex of c-jun and c-fos. Binds to TPA (phorbol ester) responsive element in DNA. Bcl-2: Protein found in mitochondrial, endoplasmic reticulum, and nuclear envelope membranes. Helps protect cell from apoptosis by inhibiting caspase (protease) activity. Bone sialoprotein: Protein found in mineralized tissue; may help in formation of hydroxylapatite. c-fos: Cellular proto-oncogene protein product of c-fos gene; protein is produced rapidly after growth factor stimulation. Phosphorylation by MAPK stabilizes c-fos. c-jun: Cellular proto-oncogene protein product of c-jun gene; forms AP-1 with c-fos. c-src: Cellular proto-oncogene protein product of c-src gene; is a tyrosine kinase important in transmitting integrin signals; these kinases phosphorylate tyrosine residues. Protein is associated with cytoplasmic face of plasma membrane. The src family of kinases includes Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes, and Yrk. Cytokine: Small secreted proteins; important in mediating immune system and inflammation. E-cadherin: Transmembrane protein used in epithelial cell cell adhesion. Found near focal adhesion sites. Factor X: Thrombokinase; important in clotting (coagulation) cascade. Requires vitamin K for synthesis. FAK: Focal adhesion kinase; a protein tyrosine kinase. Localized at focal adhesion sites by C-terminal region; associates with and is phosphorylated by c-src.

22 Special Focus: Cell-to-Cell CommunicationCell Signaling Fibrillin: ECM glycoprotein found in microfibrils; important in tissue elasticity. Fibrinogen: Blood glycoprotein which is cleaved by thrombin to form fibrin, which is found in blood clots. Fibronectin: ECM glycoprotein; important in cell migration and wound healing. Hantavirus: Negative-sense, single-stranded RNA virus in Bunyaviridae family; causes infectious respiratory disease. Herpesvirus: Double-stranded DNA virus (Herpesviridae); includes oral and genital herpes, and Epstein-Barr virus. ICAM: Intercellular adhesion molecule with structure similar to immunoglobulins (immunoglobulin super family); different forms are tissue related. Laminin: Basement membrane glycoprotein, which is a heterotrimer of α-, β-, and γ-polypeptides; forms a meshlike network. LAP-TGFβ: Amino terminal latency associated protein (LAP) fused with cytokine transforming growth factor β; is secreted into ECM. LDV region: Leucine aspartate valine sequence found in integrin ligands such as VCAM. MadCAM: Cell adhesion molecule found in mucosal cells. MAPK/ERK: Mitogen-activated protein kinase/extracellular signal-regulated kinase; a family of serine/threonine kinases, which are phosphorylated by other kinases for full activity. Important in gene transcription and regulation. Related proteins include SAPK, JNK, and p38 MAPK. Paxillin: Protein (signal transduction adaptor) that localizes to focal adhesion sites. Paxillin phosphorylation controls cell migration. PDGF: Platelet-derived growth factor; a dimeric protein. PDGF receptor is tyrosine kinase. PDGF regulates cell growth, especially angiogenesis. PECAM: Platelet/endothelial cell adhesion molecule; aids in leuckocyte migration through endothelial cell intercellular junctions. PKA: Protein kinase A or cyclic AMP-dependent protein kinase; is a serine/threonine kinase. PKA activity is high when camp levels are high. Important in glycogen and lipid metabolism. 18

23 Cell Linkages: Integrins Rho: Family of GTP-binding proteins (GTPases); members include Rho, Rac, and Cdc42. GTP-bound Rho regulated cell proliferation, actin polymerization, and intercellular adhesion. Shigella: Gram-negative, rod-shaped bacteria. Agent of shigellosis and dysentery, which are intestinal infections. Staphlococcus aureus: Gram-positive, spherical bacteria that is found on skin. Agent of pneumonia, toxic-shock syndrome, impetigo, and septicemia. Streptococcus: Gram-positive, spherical bacteria. Agent of strep throat, scarlet fever, and rheumatic fever. Talin: Protein found in focal adhesion sites; helps anchor actin to integrins and activates integrin αiibβ3. Contains binding sites for vinculin. Tenascin: ECM glycoprotein; found in areas of cell proliferation and cell migration. Tenascin levels are increased by TGF-β. Tensin: Actin-binding protein present in focal adhesion sites. Thrombospondin: Family of secreted glycoproteins. Thrombospondin-1 inhibits angiogenesis. VCAM: Vascular adhesion molecule; member of immunoglobulin super family. Mediates leukocyte-endothelial cell adhesion. Expressed on endothelial cells after cytokine stimulation. VEGF: Vascular endothelial growth factor/vascular permeability factor; increases endothelial cell proliferation and promotes angiogenesis. Vinculin: Focal adhesion protein that binds to talin or α-actinin. Vitronectin: Also called S-protein; found in ECM and blood. Interacts with collagen and important in blood clotting. Von Willebrand factor: Blood glycoprotein used in blood coagulation; binds to platelets and to collagen. 19

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25 AP Biology Free-Response Questions and Scoring Rubrics Julia Eichman Missouri Southern State University Joplin, Missouri Question Topic: Plant Reproduction 1985 Exam Question Seeds that are randomly positioned when planted in a pot of soil placed on a windowsill produce seedlings with downward growing roots and upward growing shoots. Above ground, the shoots are oriented toward light. Describe the physiological mechanisms that occur to produce: a) the downward growth of the roots b) the upward growth of the shoots c) the bending of the shoots toward light Reader s Scoring Rubric Standards: Not More Than 15 Total Points Were Given. One Point for Each of the Following: The hormone involved is auxin. In vertical roots or stems, auxin is uniformly distributed. In horizontally placed roots, auxin accumulates on the lower side. The accumulation of auxin on the lower side in roots inhibits cell elongation in the area. In horizontally placed stems, auxin accumulates on the lower side. Accumulation of auxin in stems is stimulatory.

26 Special Focus: Cell-to-Cell CommunicationCell Signaling In a laterally illuminated stem, auxin accumulated on the shady side. There is lateral transport of auxin from the sunny to the shady side, or from top to bottom in horizontally placed stems and roots. Two Points for Each of the Following: Auxin is produced in the stem apex. Auxin causes cell elongation in stems. The optimum for root growth is an amount much less than for stem growth. In high concentration, auxin is inhibitory in both stems and roots. Lateral movement of auxin requires energy. Auxin movement is too fast to be explained by diffusion. The perception of auxin in stem tips is light promoted (carotenes or flavenes). Discussion of the perception of gravity. Evidence that the site of perception is the tip. Five Points for the Following: The downward growth of roots: The geotropic response of the root is dependent on the production of a growth inhibitor or inhibitors produced in the root cap. The inhibitor(s) move from the cap through the apex to the elongating cells. If the root is horizontal, a large part of the substance is transported laterally to the lower side. The difference in concentration produces unequal growth. [T]he lower side is more inhibited and the root therefore turns down. Question Topic: Plant Reproduction 1984 Exam Question Define the following plant responses and explain the mechanism of control for each. Cite experimental evidence as part of your discussion. a) Phototropism b) Photoperiodism 22

27 AP Biology Free-Response Questions and Scoring Rubrics Reader s Scoring Rubric Standards: Phototropism: Max. = 9 points if experimental evidence is given Max. = 7 points if experimental evidence is lacking Definition: Movement in response to light (involving growth) 2 points Possibility of negative response Mechanism Auxins Distribution (apex -> stem or lateral) Elongation of cells Stem tip or coleoptile Evidence (2 points for any of the following) Darwin covered coleoptiles Paal cut coleoptiles agar, uneven placement Boysen-Jensen mica Went bioassay Photoperiodism Max. = 9 points if experimental evidence is given Max. = 7 points if experimental evidence is lacking Definition response to light/dark periods Flowering (or other response) Mechanism Categories of plants (LDP, SDP) Receptor in leaf LDP (if night shorter than minimum) SDP (if night longer than minimum) Night not day Existence of phytochrome in two forms PFR/PR interconvertible PFR active form Ratio (PR/PFR) important Possible hormonal involvement 23

28 Special Focus: Cell-to-Cell CommunicationCell Signaling Evidence Light flash in dark Grafting Ratio of PR/PFR Question Topic: Reproduction 1985 Exam Question Describe the structure of a bean seed and discuss its germination to the seedling stage. Include in your essay hormonal controls, structural changes, and tissue differentiation. Reader s Scoring Rubric Standards: Structure: Max. = 8 points Seed coat (protection) Embryo (new plant) Cotyledons (store food) Epicotyl (new shoot) Hypocotyl (new stem/root) Radicle (1st root) Plumule (1st leaves) Hilum scar (attachment) Micropyle (pollen tube entry) Germination Discussion: Max. = 12 points Imbibition of water (increases metabolism) Correct temperature (enzymes) Oxygen (for respiration) Radicle emerges first (establishes root) Subsequent shoot (photosynthesis when stored food gone) Formation of hook/arch (pulls epicotyl) Epigeal germination a. Hormonal Control Auxin in geotropism (+ or -) 24

29 AP Biology Free-Response Questions and Scoring Rubrics More auxin, lower 1/2 axis Stem/root affected differently Gibberellins stimulate length growth Cytokinins stimulate cell division Abscisic acid inhibits root cell elongation b. Structural Changes (Note: Some germination discussion is structural change.) Formation of root cap Dropping spent cotyledons Change, dark-to-light growth Branch root production Leaf primordia Two different foliage leaves c. Tissue differentiation Cell division, elongation, maturation Xylem, phloem (elaboration) Apical meristem Protoderm, ground meristem, procambium Several vascular strands, stem; one, roots Collenchyma, sclerenchyma Mesophyll, epidermis, guard cells Endodermis pericycle Root hair formation Question Topic: Flight or Flight Response 1992 Exam Question Survival depends on the ability of an organism to respond to changes in its environment. Some plants flower in response to changes in day length. Some mammals may run or fight when frightened. For both of these examples, describe the physiological mechanisms involved in the response. 25

30 Special Focus: Cell-to-Cell CommunicationCell Signaling Reader s Scoring Rubric Adaptive Turn on needed systems/turn off those not needed; understanding of acute versus chronic response, above and beyond statements in the question. Mechanism Description of nerve pathway (sensory associative motor) Sympathetic nervous system (autonomic) activation Sympathetic system innervates adrenal medulla Inhibition of parasympathetic by sympathetic Parasympathetic counters sympathetic, return to normal homeostasis; acetylcholine = neurotransmitter Epinephrine adrenalin (cause and effect) Norepinephrine noradrenalin (cause and effect) Source adrenalin from adrenal medulla (gland) Source noradrenalin from adrenal medulla and/or sympathetic nerve endings Receptor molecules on cell membranes Use of camp (second messenger) to elicit intracellular response Brief versus sustained contrasted (initial = sympathetic versus long = adrenal) Chemical structure of adrenalin/noradrenalin Effect: Max. = 7 points a. target tissues and effects (2 points) b. pupillary muscles of eye dilates pupils c. inhibits salivation d. bronchi of lungs relaxes e. increases respiratory rate f. heart muscle accelerates pulse, strengthens contraction g. piloerection muscles attached to hair follicles h. liver breaks down glycogen, stimulates release of glucose i. digestive tract decreases digestive activities peristalsis j. stomach, small intestine, pancreas inhibits secretion of digestive enzymes k. stimulates release of fatty acids from fat cells 26

31 AP Biology Free-Response Questions and Scoring Rubrics l. peripheral circulation vessels constrict m. inhibit sex structures n. relax bladder/bowels o. decreased sensation of pain p. superhuman Question Topic: Biological Recognition 1992 Exam Question Biological recognition is important in many processes at the molecular, cellular, and organismal levels. Select three of the following, and for each of the three that you have chosen, explain how the process of recognition occurs and give an example. a. Organisms recognize others as members of their own species. b. Neurotransmitters are recognized in the synapse. c. Antigens trigger antibody responses. d. Nucleic acids are complementary. e. Target cells respond to specific hormones. Reader s Scoring Rubric Standards: Four points maximum for each of the following a. Organisms recognize others as members of their own species. Definition (1 point) Importance of species recognition/definition of species/reproductive isolation prezygotic (3 points) Mechanisms (2 points) Visual/auditory/chemical/tactile/(multiple/ritual/behavioral) Recognition is innate or learned (imprinting) (1 point) Example (1 point) Visual birds, fruit flies Auditory birds, whales, frogs, insects Chemical moths, voles Tactile fruit flies, octopods Multiple albatross, butterflies, fruit flies, people, dove Imprinting ducks, goats 27

32 Special Focus: Cell-to-Cell CommunicationCell Signaling b. Neurotransmitters are recognized in the synapse. Definition (1 point) Neurotransmitter is a chemical messenger Synapse definition Mechanisms (1 point each) Neurotransmitter binds to receptor on postsynaptic membrane Receptor is a protein Lock and Key Concept (3 points) Enzymatic recognition and degradation of Neurotransmitter Reabsorption of Neurotransmitter by presynaptic membrane Presynaptic/Postsynaptic Events (1 point for any one) Stimulus (impulse, depolarization, signal, action potential) travels from presynaptic membrane (axon terminus, synaptic knob) Membrane channels opened (calcium channels, ion channels, calcium goes in) Neurotransmitter released from presynaptic neuron (synaptic vesicle) Neurotransmitter diffuses across synapse/synaptic cleft Neurotransmitter binding alters permeability Depolarizes and/or hyperpolarizes postsynaptic membrane (creates EPSP [excitatory postsynaptic potential]/creates IPSP [inhibitory postsynaptic potential]) Change membrane potential (toward or away from threshold) Opening ion channels Alter metabolism inside postsynaptic cell (2nd messenger, camp) Reversible binding of Neurotransmitter Examples (1 point) Acetylcholine (ACh)Synapse Types GABAAcetylcholinesterase (AChE) NorepinephrineCatecholamines, L-dopa Dopamine and SerotoninBiogenic Amines Endorphins/EnkephalinsNeuropeptides c. Antigens trigger antibody response Definitions (1 point for either) Antigen (Ag)foreign substance/nonself 28

33 AP Biology Free-Response Questions and Scoring Rubrics Antibody (Ab)defensive protein produced in response to Ag structure (two heavy and two light polypeptide chains) Processes (1 point for each) Selection of B cell highly specific B cell surface Ab binds Ag to activate B cellplasma cell and memory cell clones Secondary response description Ag-Ab complexamino acid sequence of light and heavy chains of hypervariable regions at N-terminus Specific site of Ag binding with Ab (Ab binding with Ag) Receptors on B cells and capping Free Ag with Ab T-cell dependent activation of B cellsmacrophage (Ag presenting cell) activates Interleukins to activate Helper T cells and B cells Generation of Ab diversity Examples of Antigens or Resultant Antibodies (1 point) IgG, IgM, IgA, IgD, IgE Bacterial cells, viruses, fungi, protozoa, allergens (pollen, dust, dander), grafts (HLA), Heterologous Ag (RBCs), Self Antigens d. Nucleic acids are complementary. Definitions (1 point) DNA and RNA are nucleic acids Nucleic acids are polymers of nucleotides Nucleotide = sugar (deoxyribose and ribose), phosphate, nitrogenous base Mechanisms (1 point for each) A with T or U, C with G or Chargaff s Rules Pyrimidine with Purine or Single ring with Double ring 2 Hydrogen Bonds with A+T/U and 3 Hydrogen Bonds with G+C or H bonds Antiparallel orientation 5'---3'/3'---5' Template requirement or semiconservative replication mechanism Primers 29

34 Special Focus: Cell-to-Cell CommunicationCell Signaling DNA/RNA polymerase requirements Elongation/Initiation Factors Divalent Cations Examples (1 point) Replication of DNA (2 strands of dsdna are complementary) Transcription of DNA into mrna, trna, rrna Translation - mrna-trna (codon/anticodon complementarity) Hybridization - DNA-DNA/DNA-RNA/Probes e. Target cells respond to specific hormones. Definition (1 point for each) Hormonechemical messenger released to travel to cause specific biological response within organism, effective at low concentration Protein hormone/receptor at cell surface (doesn t get in) Steroid hormone/receptor inside cell (does get in) Recognition of hormone is to specific receptor (specific proteins) Protein hormone involves second messenger (camp, etc.) Steroid hormone affects transcription Examples (1 point each) Any hormone/target or effect (no pheromones, allomones, attractants) Question Topic: Membranes 1993 Exam Question Membranes are important structural features of cells. (a) Describe how membrane structure is related to the transport of materials across a membrane. (b) Describe the role of membranes in the synthesis of ATP in either respiration or photosynthesis. Reader s Scoring Rubric Membranes serve diverse functions in eukaryotic and prokaryotic cells. One important role is to regulate the movement of materials into and out of cells. The phospholipid bilayer structure (fluid mosaic model) with specific membrane proteins accounts for the selective permeability of the membrane and passive and active transport 30

35 AP Biology Free-Response Questions and Scoring Rubrics mechanisms. In addition, membranes in prokaryotes and in the mitochondria and chloroplasts of eukaryotes facilitate the synthesis of ATP through chemiosmosis. Part A. (6 Maximum) Membrane Structure (3 Internal Maximum) Phospholipid structurehydrophilic, hydrophobic, amphipathic Phospholipid bilayer/fluid mosaic description Proteins embedded in the membrane Sterols embedded in the membrane Well-labeled diagram may replace one of the above. Membrane Transport (3 Internal Maximum) Use of the term selectively permeable, a good definition of selective permeability, or an explanation of the role of phospholipids or proteins, including nuclear pore proteins, in determining selective permeability. Description of the effect of size, charge, polarity, lipid solubility on membrane permeability. Mechanisms + Description Related to Structure: Passive transport: diffusion/osmosis + reference to membrane gradient Ion channel: transport as a mechanism for a change in permeability Facilitated diffusion: description (symport, antiport, uniport) Active transport: description Exocytosis, endocytosis, phagocytosis, pinocytosis: description (1 additional point) A good example of one of the above mechanisms PART B. Role of the Membrane in the Production of ATP in Photosynthesis or Respiration (6 Maximum) Chemiosmosis: Involved molecules are embedded in the membrane. Electron carriers are sequentially organized. The energy comes from the flow of electrons. H+ / Proton / ph gradient established Movement through the membrane generates ATP. A specific protein makes ATP. 31

36 Special Focus: Cell-to-Cell CommunicationCell Signaling RESPIRATION or Site is the mitochondrion Inner mitochondrial membrane (cristae) are involved in ejkaryotes Folded membrane present Cell membrane is involved in membranes prokaryotes Correct direction of H+ flow PHOTOSYNTHESIS Site is the chloroplast Thylakoid/grana are involved in eukaryotes Folded membrane present Thylakoid/grana involved in prokaryotes Correct direction of H+ flow Question Topic: Cellular Communication 1999 Exam Question Communication occurs among the cells in a multicellular organism. Choose THREE of the following examples of cell-to-cell communication, and for each example, describe the communication that occurs and the types of responses that result from this communication. Communication between two plant cells Communication between two immune-system cells Communication either between a neuron and another neuron or between a neuron and a muscle cell Communication between a specific endocrine-gland cell and its target cell Reader s Scoring Rubric Overview of point distribution: Communication between two plant cells (Max. = 4 points) Source (Max. = 1 point) Hormone-producing cell (generic) Plasmodesmata (elab. pt. for good description) Signal (Max. = 1 point) A specific plant hormone Responses/Elab. (Max. = 2 points) Various physiological changes Ion movement; H 2 0 movement; RNA movement 32

37 AP Biology Free-Response Questions and Scoring Rubrics Communication between two immune system cells (Max. = 4 points) Source (Max. = 1 point) Any two immune system cells interacting or An immune system cell interacting with the product of another immune system cell Signal (Max. = 1 point) Tc/APC docking Antibody Histamine Interferon Responses/Elaboration (Max. = 2 points) Discharge of perform; phagocytosis of pathogen; inflammatory response; phagocyte activation; Ab secretion; clonal selection Communication between two neurons or between a neuron and a muscle cell (Max. = 4 points) Source (Max. = 1 point) Sending neuron Signal (Max. = 1 point) Neurotransmitter Responses/Elaboration (Max. = 2 points) Neuron-neuron: Chemical gating; depolarization of postsynaptic membrane; EPSP, IPSP, or both Neuron-muscle: Action potential to T tubules; Ca ++ release from sarcoplasmic reticulum; Ca ++ binding to troponin; cross-bridge formation Communication between a specific endocrine-gland cell and its target cell (Max. = 4 points) Source (Max. = 1 point) Specific gland (elaboration point for peptide vs. steroid hormone pathways) Signal (Max. = 1 point) Specific hormone Responses/Elaboration (Max. = 2 points) Specific effect 33

38 Special Focus: Cell-to-Cell CommunicationCell Signaling Question Topic: Proteins 2001 Exam Question Proteinslarge complex moleculesare major building blocks of all living organisms. Discuss the following in relation to proteins. a. The chemical composition and levels of structure of proteins b. The roles of DNA and RNA in protein synthesis c. The roles of proteins in membrane structure and transport of molecules across the membrane Reader s Scoring Rubric a. Chemical composition (Max. = 2 points) Amino acids are the basic building blocks of proteins (Max. = 1 point) Amino acids contain amino, carboxyl, and R groups or correct structural formula showing amino, carboxyl, and R group attached to central carbon or proteins are composed of carbon, hydrogen, oxygen, and nitrogen (Max. = 1 point) R group determines the identity/properties of the amino acid (Max. = 1 point) Elaboration (Max. = 1 point) Describe addition of lipids, carbohydrates, and/or prosthetic group Levels of structure (Max. = 3 points) (Note: To obtain any points, response must name level or list in correct order.) Primary structure (Max. = 1 point) sequence (chain, string) of amino acids or the number and order of amino acids amino acids linked by peptide bonds amino acids bonded through dehydration synthesis Secondary structure (Max. = 1 point) helix and/or pleated sheet hydrogen bonds (between carboxyl and amino groups) 34